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Glycoconjugate Journal

, Volume 25, Issue 8, pp 797–802 | Cite as

Defining substrate interactions with calreticulin: an isothermal titration calorimetric study

  • Garima Gupta
  • Emiliano Gemma
  • Stefan Oscarson
  • Avadhesha Surolia
Article

Abstract

Calreticulin (CRT) is a soluble, lectin chaperone found in the endoplasmic reticulum of eukaryotes. It binds the N-glycosylated polypeptides via the glycan intermediate Glc1Man5–9GlcNAc2, present on the target glycoproteins. Earlier we have studied interactions of substrate with CRT by isothermal titration calorimetry (ITC) and molecular modeling, to establish that CRT recognizes the Glcα1–3 linkage and forms contacts with each saccharide moiety of the oligosaccharide Glcα1–3Manα1–2Manα1–2Man. We also delineated the amino acid residues in the sugar binding pocket of CRT that play a crucial role in sugar–CRT binding. Here, we have used mono-deoxy analogues of the trisaccharide unit Glcα1–3Manα1–2Man to determine the role of various hydroxyl groups of the sugar substrate in sugar–CRT interactions. Using the thermodynamic data obtained by ITC with these analogues we demonstrate that the 3-OH group of Glc1 plays an important role in sugar–CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. This study sheds light further on the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT.

Keywords

Calreticulin Isothermal titration calorimetry Sugar–CRT interaction 

Abbreviations

ER

Endoplasmic Reticulum

CRT

Calreticulin

CNX

Calnexin

ERAD

Endoplasmic Reticulum Associated protein Degradation

ITC

Isothermal Titration Calorimetry

GST

Gluathione-S-Transferase

IPTG

Isopropyl β-d-1-thiogalactopyranoside

MOPS

3-(N-Morpholino)-propanesulfonic acid

SDS-PAGE

Sodium dodecyl sulphate-Polyacrylamide Gel Electrophoresis

Notes

Acknowledgements

This investigation was supported by grants from the Department of Science and Technology; Government of India to A.S. GG thanks the Council for Scientific and Industrial Research, India, for the award of a Senior Research Fellowship.

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Garima Gupta
    • 1
  • Emiliano Gemma
    • 2
  • Stefan Oscarson
    • 2
    • 3
  • Avadhesha Surolia
    • 1
    • 4
  1. 1.Molecular Biophysics UnitIndian Institute of ScienceBangaloreIndia
  2. 2.Department of Organic Chemistry, Arrhenius LaboratoryStockholm UniversityStockholmSweden
  3. 3.School of Chemistry and Chemical BiologyUniversity College DublinDublinIreland
  4. 4.National Institute of ImmunologyNew DelhiIndia

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